The field of the present invention is the area of cellulolytic enzymes, nucleotide sequences encoding them and recombinant host cells and methods for producing them.
Cellulosic biomass, photosynthesized by solar energy with CO.sub.2 and H.sub.2 O, is one of the most important renewable energy resources on earth. Its effective utilization through biological processes is one approach to overcoming the shortage of foods, feeds and fuels, expected as a consequence of the explosive increase in human population [Ohmiya et al. (1997) Biotechnol. Gen. Engineer. Rev. 14:365-414]. Several types of enzymes are required for complete hydrolysis of cellulose to glucose, including endoglucanase, exoglucanase or cellobiohydrolase and .beta.-glucosidase [Filho (1996) Can. J. Microbiol. 42:1-5].
.beta.-Glucosidase (.beta.-D-glucoside glucohydrolase; EC 3.2.1.21) is common among plants, fungi and bacteria. .beta.-Glucosidase has aroused considerable interest primarily because of its involvement in the biological conversion of cellulosic material. The enzymatic saccharification of cellulosic materials to D-glucose is known to require the synergistic action of three classes of enzymes: endo-1,4-.beta.-D-glucanohydrolase (EC 3.2.1.74), 1,4-.beta.-D-cellobiohydrolase (EC 3.22.1.91), and 1,4-.beta.-D-glucosidase (.beta.-glucosidase; EC 3.2.1.21). Endo-1,4-.beta.-D-glucanases act randomly on cellulose chains, whereas 1,4-.beta.-D-cellobiohydrolases cleave cellobiosyl residues from the ends of cellulose chains, generating cellobiose as the main product. .beta.-Glucosidase acts to liberate D-glucose units from cellobiose, cello-oligosaccharides, and other glucosides [Freer (1993) J. Biol. Chem. 268:9337-9342].
Anaerobic fungi have been isolated from the alimentary tracts of herbivores and other environments [Li et al. (1997) Appl. Environ. Microbiol. 63:628-635; Wubah and Kim (1994) Abstracts of the 94.sup.th Gen. Meet. of the American Society for Microbiology. Las Vegas, Nev., USA]. They produce highly active hydrolytic enzymes [Borneman et al. (1989) Appl. Environ. Microbiol. 55:1066-1073]. Genes encoding several cellulases and xylanases have been cloned and sequenced from anaerobic fungi Neocallimastix patriciarum [Black et al. (1994) Biochem. J. 299:381-387; Denman et al. (1996) Appl. Environ. Microbiol. 62:1889-1896; Gilbert et al. (1992) Mol. Microbiol. 6:2065-2072; Zhou et al. (1994) Biochem. J. 297:359-364], Piromyces sp. [Fanuti et al. (1995) J. Biol. Chem. 270:29314-29322] and Orpinomyces sp. [Chen et al. (1998) FEMS Microbiol. Letts. 159:63-68; Li et al. (1997) Appl. Environ. Microbiol. 63:628-635]. In addition, genes coding for three mannanases from Piromyces sp. [Fanutti et al. (1995) J. Biol. Chem. 270:29314-29322; Millward-Sadler et al. (1996) FEMS Microbiol. Lett. 141:183-188] and one 1,3-1,4-.beta.-D-glucanase from Orpinomyces sp. [Chen et al. (1997) J. Bacteriol. 179:6028-6034] have been cloned and sequenced. However, genes coding for .beta.-glucosidases of anaerobic fungi have not been reported even though several such enzymes from Neocallimastix [Herbaud and Fevre (1990) Appl. Environ. Microbiol. 56:3164-3169; Li and Calza (1991 A) Enzyme Microb. Technol. 13:622-628; Li and Calza (1991B) Biochem. Biophys. Acta 1080:148-154], Orpinomyces [Chen et al. (1994) Appl. Environ. Microbiol. 60:64-70], and Piromyces [Teunissen et al. (1992) Arch. Microbiol. 158:276-281] have been purified and characterized.
There is a longfelt need in the art for .beta.-glucosidase enzymes with catalytic properties which allow for improved saccharification of cellulosic materials and partial breakdown products thereof